Signal Amplification for Cell-Free Biosensors, an Analog-to-Digital Converter

Toehold
switches are biosensors useful for the detection
of endogenous
and environmental RNAs. They have been successfully engineered to
detect virus RNAs in cell-free gene expression reactions. Their inherent
sequence programmability makes engineering a fast and predictable
process. Despite improvements in the design, toehold switches suffer
from leaky translation in the OFF state, which compromises the fold
change and sensitivity of the biosensor. To address this, we constructed
and tested signal amplification circuits for three toehold switches
triggered by Dengue and SARS-CoV-2 RNAs and an artificial RNA. The
serine integrase circuit efficiently contained leakage, boosted the
expression fold change from OFF to ON, and decreased the detection
limit of the switches by 3–4 orders of magnitude. Ultimately,
the integrase circuit converted the analog switches’ signals
into digital-like output. The circuit is broadly useful for biosensors
and eliminates the hard work of designing and testing multiple switches
to find the best possible performer.